Cerebral cavernous malformation (CCM) is a cerebrovascular disease that affects over 200,000 individuals in the US and is a common cause of stroke and seizure in young individuals. There are presently no medical therapies for CCM disease, and treatment is limited to surgical resection and anti-seizure medications. We have recently demonstrated that CCMs arise due to a gain of MEKK3 signaling and increased expression of the KLF2 and KLF4 transcription factors in CCM-deficient brain endothelial cells (ECs). These studies reveal the downstream signaling mechanism impacted in CCM disease, but they do not identify strong therapeutic targets or address the question of what activates MEKK3 in brain ECs. Our preliminary studies demonstrate that TLR4 is a critical upstream activator of endothelial MEKK3-KLF signaling that is required for CCM formation, and that gram negative bacteria (GNB) in the gut microbiome are the key in vivo ligand for this pathway in the mouse neonatal CCM model. Genetic analysis of human CCM patients by our co-investigator Dr. Helen Kim has independently identified TLR4 and its co-receptor CD14 as genes associated with CCM lesion number, suggesting that our mouse findings extend to human CCM disease. This proposal will further test the role of endothelial TLR4 signaling and the gut microbiome in CCM disease pathogenesis using mouse and human studies. Aim 1 will (i) test the role of TLR4 during CCM formation in adult mice, (ii) determine whether blocking ant-TLR4 and anti-CD14 antibodies prevent CCM formation in the mouse model, (iii) use mouse genetic approaches to determine the pathway by which TLR4 activates MEKK3 during CCM formation, and (iv) apply genetic approaches to fully interrogate the TLR4-MEKK3- KLF pathway in human patients with CCM disease due to a common mutation in KRIT1. Aim 2 will test the role of GNB and the gut microbiome in CCM pathogenesis by (i) determining whether CCM lesions arise in germ-free animals, (ii) defining microbiomes that confer resistance or susceptibility to CCM disease in mice, and (iii) testing whether breakdown of the gut epithelial barrier accelerates CCM formation in mice. These studies will significantly expand our understanding of the molecular and physiologic basis of CCM pathogenesis, and lay the foundation for translating these discoveries to new therapies for CCM disease based on blocking TLR4 signaling (for which FDA approved agents already exist) or altering the gut microbiome.